title: Mechanistic consequences of replacing the active-site nucleophile Glu-358 in Agrobacterium sp. beta-glucosidase with a cysteine residue.
abstract: Retaining glycosidases achieve the hydrolysis of glycosidic bonds through the assistance of two key active-site carboxyls. One carboxyl functions as a nucleophile/leaving group, and the other acts as the acid-base catalyst. It has been suggested that a cysteine residue could fulfil the role of the active site nucleophile [Hardy and Poteete (1991) Biochemistry 30, 9457-9463]. To test the validity of this proposal, a kinetic evaluation was conducted on the active-site nucleophile cysteine mutant (Glu-358-->Cys) of the retaining beta-glucosidase from Agrobacterium sp. The Glu-358-->Cys mutant was able to complete the first step (glycosylation) of the enzymic mechanism, forming a covalent glycosyl-enzyme intermediate, but the rate constant for this step was decreased to 1/10(6) of that of the native enzyme. The subsequent hydrolysis (deglycosylation) step was also severely affected by the replacement of Glu-358 with a cysteine residue, with the rate constant being depressed to 1/10(7) or less. Thus Cys-358 functions inefficiently in both the capacity of catalytic nucleophile and leaving group. On the basis of these results it seems unlikely that the role of the active-site nucleophile in retaining glycosidases could successfully be filled by a cysteine residue.
authors.affiliation: Protein Engineering Network of Centres of Excellence and the Departments of Chemistry and Microbiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
authors.forename: S L
authors.initials: SL
authors.lastname: Lawson
authors.forename: R A
authors.initials: RA
authors.lastname: Warren
authors.forename: S G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine; D005971:Glutamates; D007700:Kinetics; D013058:Mass Spectrometry; D011489:Protein Denaturation; D012231:Rhizobium / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014508:Urea; D001617:beta-Glucosidase / Q000737:chemistry*
chemical_list: D005971:Glutamates; D013438:Sulfhydryl Compounds; D014508:Urea; D001617:beta-Glucosidase; D003545:Cysteine